The tumor targeting performance of anti-CD166 Probody drug conjugate CX-2009 and its parental derivatives as monitored by 89Zr-immuno-PET in xenograft bearing mice.

Probody® therapeutics are recombinant masked monoclonal antibody (mAb) prodrugs that become activated by proteases present in the tumor microenvironment. This makes them attractive for use as Probody drug conjugates (PDCs). CX-2009 is a novel PDC with the toxic drug DM4 coupled to an anti-CD166 Probody therapeutic. CD166 is overexpressed in multiple tumor types and to a lesser extent by healthy tissue. Methods: The tumor targeting potential of CX-2009 was assessed by performing 89Zr-immuno-PET/biodistribution/therapy studies in a CD166-positive H292 lung cancer mouse model. Head-to-head comparisons of CX-2009 with the Probody therapeutic without DM4 (CX-191), the unmasked antibody drug conjugate (ADC) CX-1031, and the parental mAb CX-090 were performed. All constructs were 89Zr labeled in a GMP compliant way, administered at 10, 110, or 510 µg, and ex vivo biodistribution was assessed at 24, 72, and 168 hours post-injection. Results: Comparable biodistribution was observed for all constructs, confirmed with PET/CT. Tumors showed the highest uptake: 21.8 ± 2.3 ([89Zr]Zr-CX-2009), 21.8 ± 5.0 ([89Zr]Zr­CX-191), 18.7 ± 2.5 ([89Zr]Zr-CX-1031) and 20.8 ± 0.9 %ID/g ([89Zr]Zr-CX-090) at 110 µg injected. Increasing the dose to 510 µg resulted in lower tumor uptake and higher blood levels for all constructs, suggesting receptor saturation. In addition, CX-2009 and CX-1031 showed similar therapeutic potential. Conclusions: CX-2009 is optimally capable of targeting CD166-expressing tumors when compared with its derivatives, implying that enzymatic activation inside the tumor, required to allow CD166 binding, does not limit tumor targeting. Because CX-2009 does not bind to mouse CD166, however, reduced targeting of healthy organs should be confirmed in ongoing clinical 89Zr-immuno-PET studies.

Multifaceted effects of soluble human CD6 in experimental cancer models.

BACKGROUND: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. METHODS: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEµTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. RESULTS: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. CONCLUSIONS: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.

Allele substitution and dominance effects of CD166/ALCAM gene polymorphisms for endoparasite resistance and test-day traits in a small cattle population using logistic regression analyses.

The study investigated the effects of four single-nucleotide polymorphisms (SNPs) in the activated leukocyte cell adhesion molecule (ALCAM) gene on liver fluke (Fasciola hepatica) infections (FH-INF), gastrointestinal nematode infections (GIN-INF) and disease indicator traits [e.g. somatic cell score (SCS), fat-to-protein ratio (FPR)] in German dual-purpose cattle (DSN). A genome-wide association study inferred the chip SNP ALCAMc.73+32791A>G as a candidate for F. hepatica resistance in DSN. Because of the crucial function of ALCAM in immune responses, SNPs in the gene might influence further resistance and performance traits. Causal mutations were identified in exon 9 (ALCAMc.1017T>C) and intron 9 (ALCAMc.1104+10T>A, ALCAMc.1104+85T>C) in a selective subset of 94 DSN cows. We applied logistic regression analyses for the association between SNP genotypes with residuals for endoparasite traits (rINF-FH, rGIN-INF) and estimated breeding values (EBVs) for test-day traits. The probability of the heterozygous genotype was estimated in dependency of the target trait. Allele substitution effects for rFH-INF were significant for all four loci. The T allele of the SNPs ALCAMc.1017T>C and ALCAMc.1104+85T>C was the favourable allele when improving resistance against FH-INF. Significant allele substitution for rGIN-INF was only found for the chip SNP ALCAMc.73+32791A>G. We identified significant associations between the SNPs with EBVs for milk fat%, protein% and FPR. Dominance effects for the EBVs of test-day traits ranged from 0.00 to 0.47 SD and were in the direction of improved resistance for rFH-INF. We estimated favourable dominance effects from same genotypes for rFH-INF and FPR, but dominance effects were antagonistic between rFH-INF and SCS.

New Targeted Therapies for Uncontrolled Asthma.

Mechanistic studies have improved our understanding of molecular and cellular components involved in asthma and our ability to treat severe patients. An mAb directed against IgE (omalizumab) has become an established add-on therapy for patients with uncontrolled allergic asthma and mAbs specific for IL-5 (reslizumab, mepolizumab), IL-5R (benralizumab), and IL-4R (dupilumab) have been approved as add-on treatments for uncontrolled eosinophilic (type 2) asthma. While these medications have proven highly effective, some patients with severe allergic and/or eosinophilic asthma, as well as most patients with severe non-type-2 disease, have poorly controlled disease. Agents that have recently been evaluated in clinical trials include an antibody directed against thymic stromal lymphopoietin, small molecule antagonists to the chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) and the receptor for stem cell factor on mast cells (KIT), and a DNA enzyme directed at GATA3. Antibodies to IL-33 and its receptor, ST2, are being evaluated in ongoing clinical studies. In addition, a number of antagonists directed against other potential targets are under consideration for future trials, including IL-25, IL-6, TNF-like ligand 1A, CD6, and activated cell adhesion molecule (ALCAM). Clinical data from ongoing and future trials will be important in determining whether these new medications will offer benefits in place of or in addition to existing therapies for asthma.

Activated Leukocyte Cell Adhesion Molecule Stimulates the T-Cell Response in Allergic Asthma.

RATIONALE: The activated leukocyte cell adhesion molecule (ALCAM) is a cluster of differentiation 6 ligand that is important for stabilizing the immunological synapse and inducing T-cell activation and proliferation. OBJECTIVES: In this study, we investigated the role of ALCAM in the development of inflammation in allergic asthma. METHODS: An ovalbumin (OVA)-induced allergic asthma model was established in wild-type (WT) and ALCAM-deficient (ALCAM-/-) mice. T-cell proliferation was evaluated in cocultures with dendritic cells (DCs). Bone marrow-derived dendritic cells (BMDCs) from WT and ALCAM-/- mice were cultured and adoptively transferred to OT-II mice for either OVA sensitization or challenge. An anti-ALCAM antibody was administered to assess its therapeutic potential. ALCAM concentrations in the sputum and serum of children with asthma were quantified by ELISA. MEASUREMENTS AND MAIN RESULTS: Inflammatory responses were lower in ALCAM-/- mice than in WT mice, and T cells cocultured with DCs from ALCAM-/- mice showed reduced proliferation relative to those cocultured with DCs from WT mice. A decreased inflammatory response was observed upon adoptive transfer of BMDCs from ALCAM-/- mice as compared with that observed after transfer of BMDCs from WT mice. In addition, anti-ALCAM antibody-treated mice showed a reduced inflammatory response, and sputum and serum ALCAM concentrations were higher in children with asthma than in control subjects. CONCLUSIONS: ALCAM contributes to OVA-induced allergic asthma by stimulating T-cell activation and proliferation, suggesting it as a potential therapeutic target for allergic asthma.

Effects of complete vagotomy and blockage of cell adhesion molecules on interferon-α induced behavioral changes in mice.

OBJECTIVES: Sickness behavior and chronic immune diseases are frequently associated with depressive symptomatology. In addition, immune activation by single cytokine therapies, such as treatment of malignancies and hepatitis C with interferon-alpha (IFN-α) often induces significant changes in emotional reactivity and affect. However, underlying pathogenic mechanisms of cytokine-induced brain dysfunction largely remain unknown. METHODS: We presently demonstrate the induction of anxiety- and depressive-like behavior in male BALB/c mice after prolonged treatment with murine IFN-α for up to four weeks. Subsequently, neural and cellular communication routes between the immune system and the brain were examined. RESULTS: IFN-α induced anxious and depressive-like behavior in a light dark, open field, tail suspension, and novel object paradigm with a maximum effect after two weeks of treatment. Effect sizes of IFN-α varied between variables from 23 to 41%. Behavioral deficits were not prevented by complete vagotomy, or by blocking leukocyte function-associated-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) and activated leukocyte cellular adhesion molecule (ALCAM)-mediated cellular adhesion events, which are both involved in immune cell entry to the brain. CONCLUSIONS: We demonstrate emergence of anxiety- and depressive-like behavior in a mouse model of sustained IFN-α application allowing investigation of its pathogenic mechanisms, which is of clinical importance. We failed to demonstrate a critical effect for the vagus nerve or adhesion molecules, but current experiments do not allow excluding the vagus nerve as an afferent communication route. Future studies are needed to unveil if both pathways can be completely excluded in the etiology of behavioral deficits induced by IFN-α.

CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.

The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6-CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

Cocaine hijacks σ1 receptor to initiate induction of activated leukocyte cell adhesion molecule: implication for increased monocyte adhesion and migration in the CNS.

Human immunodeficiency virus (HIV)-associated increase in monocyte adhesion and trafficking is exacerbated by cocaine abuse. The underlying mechanisms involve cocaine-mediated upregulation of adhesion molecules with subsequent disruption of the blood-brain barrier (BBB). Recently, a novel activated leukocyte cell adhesion molecule (ALCAM) has been implicated in leukocyte transmigration across the endothelium. We now show that upregulation of ALCAM in the brain endothelium seen in HIV(+)/cocaine drug abusers paralleled increased CD68 immunostaining compared with HIV(+)/no cocaine or uninfected controls, suggesting the important role of ALCAM in promoting leukocyte infiltration across the BBB. Furthermore, ALCAM expression was increased in cocaine-treated mice with concomitant increase in monocyte adhesion and transmigration in vivo, which was ameliorated by pretreating with the neutralizing antibody to ALCAM, lending additional support to the role of ALCAM. This new concept was further confirmed by in vitro experiments. Cocaine-mediated induction of ALCAM in human brain microvascular endothelial cells through the translocation of σ receptor to the plasma membrane, followed by phosphorylation of PDGF-ß (platelet-derived growth factor-ß) receptor. Downstream activation of mitogen-activated protein kinases, Akt, and NF-κB (nuclear factor-κB) pathways resulted in induced expression of ALCAM. Functional implication of upregulated ALCAM was confirmed using cell adhesion and transmigration assays. Neutralizing antibody to ALCAM ameliorated this effect. Together, these findings implicate cocaine-mediated induction of ALCAM as a mediator of increased monocyte adhesion/transmigration into the CNS.

CD6 synergistic co-stimulation promoting proinflammatory response is modulated without interfering with the activated leucocyte cell adhesion molecule interaction.

The CD6 membrane-proximal scavenger receptor cysteine-rich domain (SRCR3) includes the activated leucocyte cell adhesion molecule (ALCAM) binding site. CD6-ALCAM mediates a low-affinity interaction and their long-term engagement contributes to the immunological synapse. Their ligation may play a dual function, facilitating stable adhesion between the antigen-presenting cells and T cells during the early activation phase and later in the proliferative phase of the immune response. This study explored the strength of the CD6 co-stimulatory effect and whether CD6 co-stimulation with its natural ligand ALCAM also contributes to the lymphocyte effector differentiation. It was found that CD6-ALCAM interaction in vitro induced a synergistic co-stimulation of normal human peripheral blood mononuclear cells, defined by Bliss analysis. CD6 co-stimulation enhanced the CD3 proliferative efficacy by 23-34%. Moreover, a fivefold increment in the CD25 molecules number with a distinct gene transcription profile associated with cell activation, differentiation, survival and adhesion molecules was observed over CD3 single activation. Additionally, CD6 co-stimulation in excess interleukin (IL)-2 promotes a preferentially proinflammatory response. Besides, a CD6 membrane-distal domain (SRCR1)-specific non-depleting monoclonal antibody (mAb) inhibited the induced proliferation in the presence of ALCAM, reducing interferon-γ, IL-6 and tumour necrosis factor-α production. These results suggest that CD6 co-stimulation enhances the intrinsic activity of the CD3 activation pathway and contributes to the T helper type 1 subset commitment, enhancing the IL-2 sensitivity of recent activated human lymphocytes. It supports the role of CD6 as a susceptibility gene for pathological autoimmunity leading to tissue inflammation, and its relevance for targeted therapy.

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth.

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.

Cancer vaccine with mimotopes of tumor-associated carbohydrate antigens.

The GD2 ganglioside, displayed by five carbohydrate Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glcbeta residues attached to a ceramide chain that anchors the ganglioside in the cell membrane, is expressed on neuroectodermally derived tumors. GD2 has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have generated 47-LDA mimotope of GD2 by screening a phage display peptide library with anti-GD2 mAb 14G2a and reported that vaccination with the 47-LDA mimotope elicited GD2 cross-reactive IgG antibody responses as well as MHC class I-restricted CD8(+) T cells to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated antigen cross-reacting with 47-LDA. Immunoblotting studies using 14G2a mAb demonstrated that this antibody cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional (3D) collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by 105 kDa activated leukocyte cell adhesion molecules (ALCAM/CD166). The CD166 glycoprotein was shown to be recognized by 14G2a antibody, and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166 and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate antigens.

Targeting a mimotope vaccine to activating Fcgamma receptors empowers dendritic cells to prime specific CD8+ T cell responses in tumor-bearing mice.

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Expression of human NDRG2 by myeloid dendritic cells inhibits down-regulation of activated leukocyte cell adhesion molecule (ALCAM) and contributes to maintenance of T cell stimulatory activity.

We reported previously that N-myc downstream-regulated gene 2 (NDRG2), a member of a new family of differentiation-related genes, is expressed specifically in dendritic cells (DC) differentiated from monocytes, CD34(+) progenitor cells, and the myelomonocytic leukemic cell line. In this study, we demonstrate that NDRG2 protein expression is detected, not only in in vitro-differentiated DC but also in primary DC from lymph nodes, thymus, and skin when anti-NDRG2 antibodies are used. As predicted from previous studies investigating the mRNA expression pattern of several types of cell lines, progenitor cells, and DC, NDRG2 protein was expressed strongly in DC. Its expression was detected at significant levels after differentiation from progenitor cells. RNA interference of NDRG2 demonstrated that activated leukocyte cell adhesion molecule (ALCAM) expression is down-regulated specifically in DC differentiated from NDRG2 small interfering RNA (siRNA)-transfected monocytes. This was consistent with our observation that U937 cells transfected with NDRG2 became resistant to the GM-CSF/IL-4-induced ALCAM reduction. Furthermore, DC, which had differentiated from NDRG2 siRNA-transfected monocytes, showed a reduced ability to induce T cell proliferation. Taken together, our results indicate that NDRG2 is able to preserve ALCAM expression during DC differentiation from monocytes under cytokine culture conditions and that its expression helps DC maintain costimulatory signals necessary for T cell stimulation.

Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.

Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.

Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer.

We have used a naive human single-chain fragment variable (scFv) library as a source of random shape repertoire to directly probe the altered surface chemistry of tumor cells. We reported previously the identification of more than 90 internalizing phage monoclonal antibodies targeting prostate cancer cells, including those that are hormone refractory. In this report, we describe the conversion of a panel of those scFvs into full-length human immunoglobulins (IgGs) and show that tumor specificity is retained. We have further shown that antibodies isolated from a naive phage display library can nevertheless be of high affinity towards target tumor cells. In addition, full-length IgGs retain the functionality of parental scFvs including the ability to rapidly enter target cells through receptor-mediated endocytosis and thereby to mediate efficient and specific intracellular payload delivery to tumor cells. We have used recombinant IgGs to immunoprecipitate target antigens and analyzed their molecular composition by mass spectrometry. We have identified one target antigen as activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166 and have further studied tissue specificity of this internalizing ALCAM epitope by immunohistochemistry. Our study shows that cell type-specific internalizing human antibody can be readily identified from a naive phage antibody display library, characterized with regards to sequence, affinity, tissue specificity, and antigen identity, and modified genetically and chemically to generate various forms of targeted therapeutics.

Phenotypic characterization of human colorectal cancer stem cells.

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the "cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM)(high)/CD44+ epithelial cells. Tumors originated from EpCAM(high)/CD44+ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM(high)/CD44+ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76.

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 muM at 37 degrees C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5.

Expression and adhesive activity of SC1, an Ig superfamily cell adhesion molecule, in sporadic nephroblastomas of chicken.

SC1, an immunoglobulin superfamily cell adhesion molecule, is transiently expressed during avian embryogenesis by a variety of cell types. This molecule has a homophilic binding activity with SC1 itself and promotes neurite projection from embryonic neurons. However, the potential role of this molecule in pathologic tissue specimens from chickens has yet to be elucidated. In this study, we examined the expression and functional role of SC1 in the sporadic nephroblastomas of chickens. Western blot analysis showed SC1 to be recognized as approximately 100 kDa and enriched in embryonic metanephros with a lower level in the adult kidney, while it was overexpressed in nephroblastomas. Immunohistochemically, SC1 was abundantly found in the tubular epithelia and blastemal cells of embryonic metanephros. In contrast, it had almost completely disappeared in the adult kidney; parts of the distal convoluted and intermediated tubules, collecting ducts, and Bowman's capsule slightly expressed SC1. In all 32 cases of nephroblastoma, SC1 was overexpressed in most characteristic components in tumors such as neoplastic epithelia with various types of differentiation, blastemal cell condensations, and glomeruloid bodies. Primary culture cells from a nephroblastoma expressed SC1 on the cell surface, whereas cells from the adult kidney showed only weak expression. A cell aggregation assay revealed that the dissociated cells from a nephroblastoma have strong aggregation activity, which was inhibited by anti-SC1 antibody. In contrast, the self-aggregation of adult chicken kidney cells was weaker than that of the tumor and not inhibited by the antibody. These findings suggest that the expression of SC1 might play a potential role in both the structural formation of nephroblastomas, based on its adhesive activity, and normal renal development.

Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties.